Detection of a pigeon pea witches'-broom-related phytoplasma in trees of Gliricidia sepium affected by little-leaf disease in Central America

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Abstract

The polymerase chain reaction (PCR) incorporating phytoplasma-specific ribosomal RNA primer pair P1 and P7 consistently amplified a product of expected size (1·8 kb) when template DNA for PCR was extracted from leaf and shoot samples of Gliricidia sepium from Honduras exhibiting symptoms of little-leaf disease (LLD). Restriction fragment length polymorphism analysis (RFLP) of amplified rDNA products indicated that phytoplasmas detected in LLD-affected G. sepium were very similar although not identical to phytoplasmas associated with pigeon pea witches'-broom (PPWB) disease in the western Caribbean region. Phytoplasma detection in trees was enhanced by reamplification of the P1/P7-primed PCR products with nested primers PPf1 and Tint. Nested reactions enabled additional positive detections in foliage of several G. sepium trees showing only mild or no apparent LLD symptoms. Neither rDNA RFLP nor sequence analyses of 16-23 S rRNA spacer regions revealed differences among Honduran LLD phytoplasma or between these strains and phytoplasmas detected in G. sepium with LLD-like symptoms in El Salvador, Guatemala, and Nicaragua, indicating that closely related, possibly identical, pathogens presently affect this tree species in all four countries.

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